3.2 Site-specific PEGylation of FGF2 variants
We used 5kDa monomethoxy PEG-maleimide (mPEG-MAL) to specifically modify
the surface-exposed cysteine on FGF2 and the mutants, as shown inFigure 2A . Three mono-PEGylated FGF2 conjugates were generated
and named as PEG-FGF2C69A/C87A/F17C (compound5 ), PEG-FGF2C87A (compound 6 ), and
PEG-FGF2C69A/C87A/K129C (compound 7 )
(Figure 2B) . SDS-PAGE analysis confirmed the purification of
these PEGylated FGF2 conjugates. A single band corresponding to protein
with an apparent Mw of 25kDa appeared after PEGylation (Figure
2C ). After purification, the ERK1⁄2 pathway activity was measured. All
PEG-FGF2 conjugates and native FGF2 exhibited higher ERK1/2
phosphorylation activity than that of the PBS control. In addition,
compared with FGF2, compound 5 and 6 induced about
1.5- and 2- fold higher phosphorylation, and compound 7exhibited a significant reduction in phosphorylation (Figure 2
D-E ). The decreased activity of compound 7 , in which
PEGylation would occur at the engineered cysteine near the
heparin-binding region, suggests that steric hindrance of the PEG
polymers can interfere with binding between heparin and the receptor.
The observed sensitivity of the heparin-binding region suggests designs
that do not interfere with this region of the protein may result in FGF2
conjugates with increased activity retention and stability after
site-specific PEGylation.